Biosynthesis of the Pseudomonas aeruginosa Extracellular Polysaccharides, Alginate, Pel, and Psl

Abstract

Pseudomonas aeruginosa thrives in many aqueous environments and is an opportunistic pathogen that can cause both acute and chronic infections. Environmental conditions and host defenses cause differing stresses on the bacteria, and to survive in vastly different environments, P. aeruginosa must be able to adapt to its surroundings. One strategy for bacterial adaptation is to self-encapsulate with matrix material, primarily composed of secreted extracellular polysaccharides. P. aeruginosa has the genetic capacity to produce at least three secreted polysaccharides; alginate, Psl, and Pel. These polysaccharides differ in chemical structure and in their biosynthetic mechanisms. Since alginate is often associated with chronic pulmonary infections, its biosynthetic pathway is the best characterized. However, alginate is only produced by a subset of P. aeruginosa strains. Most environmental and other clinical isolates secrete either Pel or Psl. Little information is available on the biosynthesis of these polysaccharides. Here, we review the literature on the alginate biosynthetic pathway, with emphasis on recent findings describing the structure of alginate biosynthetic proteins. This information combined with the characterization of the domain architecture of proteins encoded on the Psl and Pel operons allowed us to make predictive models for the biosynthesis of these two polysaccharides. The results indicate that alginate and Pel share certain features, including some biosynthetic proteins with structurally or functionally similar properties. In contrast, Psl biosynthesis resembles the EPS/CPS capsular biosynthesis pathway of Escherichia coli, where the Psl pentameric subunits are assembled in association with an isoprenoid lipid carrier. These models and the environmental cues that cause the cells to produce predominantly one polysaccharide over the others are subjects of current investigation.

Keywords: Pel polysaccharide; Pseudomonas aeruginosa; Psl polysaccharide; Rossmann fold; alginate; glycosyltransferase.

Figure 1

Structures of Alginate and Psl polysaccharide. (A) P. aeruginosa alginate is composed of d-mannuronic acid residues interspersed with l-guluronic acid residues. The hydroxyl groups of the d-mannuronic acid residues may be O-acetylated at the C2′ and/or C3′ positions. Since modification of the d-mannuronate residues occurs at the polymer level, alginate has a random structure. Shown is an example of a possible alginate subunits arrangement, where d-mannuronate subunits are epimerized to l-guluronate or decorated with O-acetyl groups. (B) Psl polysaccharide is composed of a repeating pentamer consisting of d-mannose, l-rhamnose, and d-glucose residues.

Figure 2

Extracellular polysaccharides of P. aeruginosa, visualized using three approaches. (A) Atomic Force Microscopy image of P. aeruginosa FRD1, showing alginate as a soft loosely adhered polymer that surrounds the cells. (B) Confocal laser scanning microscopy (CLSM) image of hydrated P. aeruginosa PAO1, cultured as a pellicle. The CLSM image shows a three-dimensional reconstruction of the pellicle, with the P. aeruginosa cells expressing the green fluorescent protein and the Psl polysaccharide counterstained with CellMask Orange (Invitrogen Corp.). (C) Scanning Electron Microscopy image of P. aeruginosa PA14, cultured as a pellicle. The image shows the extracellular matrix, which includes Pel, as a fabric-like matrix that surrounds and connects the cells that form a microbial mat at the air-water interface.

Figure 3

Genetic structure of the Pel, alg, and Psl operons. The genes are color coded by proposed function as shown in Figures 4–6.

Figure 4

Proposed structure of the alginate biosynthetic complex. The proteins are color coded according to proposed function as described in Figure 3.

Figure 5

Proposed structure of the Pel biosynthetic complex. The enzymes are color coded according to proposed function as described in Figure 3.

Figure 6

Proposed structure of the Psl biosynthetic complex. The enzymes are color coded according to proposed function as described in Figure 3.

Figure 7

Atomic force microscopy images of hydrated P. aeruginosa FRD1 and FRD11131 algD::Tn501 biofilm cells, collected at 90% humidity. (A) When living at an air/water interface, FRD1 is able to maintain its shape during dehydration. (B) FRD1131 biofilm cells dry out quickly and collapses under the same conditions. Once dry, the extracellular polymer is removed from FRD1 by imaging, but the surface of the FRD1 cells is distinct from FRD1131.